Nucleotide sequence and transcriptional analysis of the polyhedrin gene of Spodoptera exigua nuclear polyhedrosis virus

Abstract

The nucleotide sequence of a 1.1 kbp fragment of the multiple nucleocapsid nuclear polyhedrosis virus (MNPV) of Spodoptera exigua (Se) containing the polyhedrin gene was determined. An open reading frame (ORF) of 738 nucleotides (nt) was detected. This ORF encoded a protein of 246 amino acids with a predicted Mr of 29K. The nucleotide and amino acid sequences were compared with the sequences of eight other NPV polyhedrins. The SeMNPV polyhedrin protein was most closely related to S. frugiperda MNPV polyhedrin with differences in only five amino acids, and most distantly related to the Lymantria dispar MNPV polyhedrin. The size of the mRNA was approximately 1000 nt, as determined by Northern blot analysis. Using primer extension assays and S1 nuclease mapping the transcriptional start and stop sites of the polyhedrin mRNA were located. The 5' regulatory sequence appeared to be 44 nt in length with the mRNA start site predominantly at the first A of the TAAG consensus start sequence. Two degenerate poly(A) signals were found immediately downstream of the translational stop signal. The transcriptional stop was located approximately 230 nt downstream from the translational stop signal, in an AT-rich sequence that appears to be common to all baculovirus polyhedrin genes. The SeMNPV polyhedrin mRNA does not appear to be polyadenylated.

Sequence and Transcriptional Analysis of the Ubiquitin Gene Cluster in the Genome of Spodoptera exigua Nucleopolyhedrovirus

“…This supports the idea that the bacutovirus genome organization has been rearranged during its evolutionary history (Gombart et al, 1989). Sequence comparisons of the SeMNPV v-ubi-xb187 gene cluster with AcMNPV and OpMNPV, added to comparisons based on polyhedrin gene (van Strien et al, 1992;Zanotto et aI., 1993) and pl0 gene (Zuidema et al, 1993) sequences, confirmed the distant relationship of SeMNPV to these two viruses.…”

“…), as described by Xie & Rothblum (1991). Total RNA was denatured, electrophoresed in agarose gels and blotted onto Hybond-N nylon membrane (van Strien et al, 1992). To identify v-ubi and ORF xb187 transcripts, the blots were hybridized for 16 h at 65 °C with [~-32P]CTP-labelled riboprobes.…”

Spodoptera exigua multicapsid nucleopolyhedrovirus deletion mutants generated in cell culture lack virulence in vivo

“…When the genetic organization of SeMNPV is compared with that of Orgyia pseudotsugata MNPV (OpMNPV) and of AcMNPV with respect to the location of the polyhedrin and pl0 genes, the SeMNPV polyhedrin gene (van Strien et al, 1992) is transcribed in the same direction as the AcMNPV polyhedrin gene (Ayres eta]., 1994), but in the opposite direction to the OpMNPV polyhedrin gene (Leisy et al, 1986a, b). The distances between these two genes in the various viruses are also different, being 19"3 kbp for AcMNPV (Ayres et al, 1994), 22"3 kbp for OpMNPV (Leisy et al, 1986a, b) and 11"6 kbp for SeMNPV, respectively (van Strien et al, 1992;Zuidema et al, 1993).…”

“…This map was further validated by hybridizing individual 32P-labelled XbaI fragments to Southern blots of SeMNPV PD-DNA digested (single and double) with the seven restriction enzymes. The adenine residue at the translational initiation codon of the polyhedrin gene, upstream of the junction between the XbaI-D and -R fragments (van Strien et al, 1992) was designated as the zero point of the physical map (Fig. 3 a, c).…”

“…Restriction fragment length polymorphism in several SeMNPV isolates has been reported (Caballero et al, 1992), but a detailed physical map of the viral genome is not yet available. Three SeMNPV genes have been characterized and provisionally localized on genome fragments: polyhedrin (van Strien et al, 1992), pl0 (Zuidema et al, 1993) andubiquitin (van Strien et al, 1996). Two insect cell lines derived from S. exigua have been described that support SeMNPV replication (Gelernter & Federici, 1986;Hara et at., 1993Hara et at., , 1994.…”